1. Field of the Invention
This invention relates to an enzyme sensor for measuring the concentration of biological substrate with an amperometric method using an enzyme electrode and, more particularly, to an enzyme sensor utilizing a reducing catalytic action of di-valent iron ions structurally introduced in a thin film of clay (i.e., montmorillonite).
More specifically, the invention concerns an enzyme sensor using an enzyme electrode and a method of manufacturing the same and, more particularly, an enzyme sensor for measuring the concentration of substance under measurement by detecting a change oxidation/reduction response current which is proportional to the quantity of oxygen (O.sub.2) consumed by enzymic reaction and a method of manufacturing the same.
2. Description of the Prior Art
Enzyme sensors are well known as biological sensors. They are utilized mainly for clinical chemical analysis, and among them are those, which are in practical use for glucose in blood, urea and neutral and phospholipids as substrate under measurement.
For example, in case of glucose as subject of measurement an enzymic reaction takes place as follows. ##STR1##
This means that .beta.-D-glucose consumes oxygen (O.sub.2) by the action of .beta.-D-glucose oxidase (GO.sub.x), thus producing organic acid (gluconolactone) and hydrogen peroxide (H.sub.2 O.sub.2). Therefore, the concentration of glucose can be measured from the quantity of generated hydrogen peroxide or gluconolactone.
When measuring the glucose concentration from the quantity of generated hydrogen peroxide, the generated hydrogen peroxide is oxidized with a metal electrode, and the oxidation current is measured. Alternatively, the generated hydrogen peroxide is reduced, and the reduction current is measured. However, the oxidation or reduction current is influenced by oxygen, or with a prior art sensor electrode it is liable to be influenced by a change in the surface state of the electrode. Further, in the principle of measurement by electrochemical method the sensor construction consists of electrode substrate, liquid, enzyme-fixed film and liquid under test, and miniaturization of the sensor is difficult due to the liquid present between the electrode and film.
There are further prior art methods of measurement, that is,
(1) one, in which hydrogen peroxide is decomposed with catalase into oxygen and water, and the quantity of resultant oxygen is measured, and
(2) one, in which iodide ions are oxidized in the presence of peroxidase or an inorganic catalyst (e.g., molybdenum), the quantity of iodine is measured by causing a reaction ##STR2## and indirectly measuring the quantity of hydrogen peroxide.
As shown above, in any prior art method of measuring the quantity of glucose from the quantity of generated hydrogen peroxide, the quantity of consumed oxygen or generated iodine is measured, and the quantity of generated hydrogen peroxide, is determined indirectly from the measured quantity.
In such method of measurement, which involves two reaction stages, an electrode for measuring oxygen or iodine is necessary in addition to an enzymic electrode for decomposing glucose or the like into hydrogen peroxide. In addition, the measurement is very cumbersome and requires a long time.
Further, since in the prior art electrochemical method the sensor includes inner liquid, contamination of the liquid under test is liable, and also miniaturization of the sensor is difficult.
Meanwhile, a sensor part of the enzyme sensor for measuring the concentration of glucose in blood through measurement of the consumption of oxygen generated in enzymic reaction mostly uses a commonly termed Clark type oxygen electrode, which uses platinum as cathode and silver/silver chloride as anode and has chloride-added standard buffer solution as sealed inner liquid and an outer cover film selectively permeable to oxygen gas.
However, the Clark type oxygen electrode can be miniaturized with difficulty. In addition, since it has an inner solution chamber, it is subject to leakage and contamination. Further, the response speed is slow. Still further, while quick response can be obtained with a separate type oxygen electrode, a cathode type platinum electrode is subject to influence of H.sup.+ ion concentration. For this reason, with a fluid system subject to great pH changes distinction with true oxygen partial pressure (PO.sub.2) concentration is difficult.